日本血吸虫CuZn-SOD基因全长cDNA的识别及真核表达载体的构建

目的　识别日本血吸虫大陆株铜锌超氧化物歧化酶 (SjCuZn SOD)基因 ,构建SjCuZn SOD的真核表达载体。方法　将曼氏血吸虫 (Sm )的CuZn SOD全长cDNA序列上网比对 ,寻找Sj相关EST。设计特异性引物从尾蚴cDNA文库扩增相应序列 ,经双酶切、连接、转化 ,克隆入 pcDNA3 .0真核表达质粒 ,并鉴定阳性克隆。　 结果　找到Sj相关EST(登录号BU794891) ,核酸阅读框 (ORF)分析和BLAST比对分析等方法判断为SjCuZn SOD完整的cDNA编码序列 ,含462bp完整阅读框 ,编码 15 4aa。经单双酶切、PCR扩增、核酸测序鉴定 ,验证成功构建了 pcDNA3 .0 SOD真核重组表达载体。　结论　成功构建真核重组表达载体 pcDNA3 .0 SOD ,为在真核表达系统研究SjCuZn SOD基因功能奠定了基础

SCHISTOSOMA JAPONICUM:SEQUENCE FINDING AND CLONING OF THE GENE ENCODING CuZn-SOD

Objective To find CuZn-SOD gene of Schistosoma japonicum(Sj)and subclone into eukaryotic expression vector. Methods Seek correlated EST of Sj by comparing CuZn-SOD complete cDNA of Schistosoma mansoni(Sm) with other sequences of shistosoma on internet. Specific primers were synthesized and used to amplify CuZn-SOD gene by PCR from Sj cercariae cDNA library, which was then subcloned into pcDNA3.0 vector. Positive clone was identified. Results Sj EST(Accession No.BU794891)was obtained from Genbank, and was analyzed by bioinformatics method. The sequence contains a 462 bp length open reading frame, which encodes 154 amino acid residues. The novel gene was identified to be CuZn-SOD .It was subcloned into pcDNA3.0 vector and identified by restriction analysis, PCR and sequencing. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.0-SOD was successfully constructed. We laid the base for further study.